We have characterized HeLa cell microtubules obtained after 4 cycles of polymerization-depolymerization and have found a major accessory protein (MAP) associated with these microtubules which has a MW equal to 201-206,000 daltons by SDS-PAGE. Further, by in vitro reconstitution experiments this MAP promotes assembly of HeLa cell tubulin to form microtubules at least as efficiently as calf brain MAP-2. Current experiments are aimed at determining if HeLa MAPs are essential for adenovirus edge-binding in vitro to microtubules. We had previously shown there was a need for brain MAP-2s in this assay. On another front we have performed a quantitative study of adenovirus binding to HeLa cell microtubules in vivo. We showed that after infection with adenovirus type 2 or 5, at 1 hour after infection, a significant portion of the virus particles observed (31%) was found to be associated with microtubules. On a random basis we would only have expected a 1 or 2% association of virus with microtubules. Currently, double immunofluorescence experiments are being performed to determine whether this early adenovirus association with microtubules can be visualized using antisera to tubulin in conjunction with antisera to virus.